Many human diseases result from improper regulation of signaling networks in cells controlled by receptors and GTPases. The focus for this proposal is to define the role of the MAPK signaling network in controlling integrated physiological responses. MAPKs are members of a three kinase cascade composed of the MAPK, MAP2K and MAP3K. MAP3Ks integrate the MAPK signaling network in response to GTPases with parallel signaling pathways for control of physiological responses. MEKK1, a MAP3K, is the only kinase within the network to encode a kinase domain and a RING domain with E3 ligase activity. MEKK1 is also the only kinase in all genomes to encode a SWIM-RING domain architecture for binding and ubiquitinating protein substrates. MEKK1 is both a kinase for phosphorylation of specific substrates and an E3 ligase with a unique SWIM-RING domain architecture for ubiquitination of specific substrates. MEKK1 regulates the composition of the AP-1 transcription complex by regulating the levels of Fra-2 and JunB. MEKK1 is also capable of targeting c-Jun for ubiquitination and degradation. Our hypothesis is that MEKK1 functions to stimulate the activity of specific MAPK pathways (primarily JNK and ERK1/2), coordinately regulate the degradation of specific target substrates (e.g., c-Jun, JunB, Fra2), and controls AP-1 composition to alter specific gene expression for the integrated control of responses including migration, invasion, wound healing, inflammation, tissue remodeling and metastasis. The proposed studies are a systems approach to define how MEKK1 functions to control complex regulatory responses in cells and animals, which we believe will lead to novel therapeutic strategies for the treatment of diseases that involve tissue remodeling. Aim I will define function for the SWIM and RING domains of MEKK1 for binding and ubiquitination of target proteins. Approaches involve the use of quantitative mass spectrometry and ubiquitination assays to define protein substrates for the MEKK1 SWIM-RING domains. Aim II will provide genomic analysis of MEKK1-regulated gene expression using Chromatin-IP (ChIP) combined with promoter tiling arrays and single molecule-based DNA sequencing. The studies will define genes with differential binding of Fra2 and/or JunB in MEKK1-/- cells versus wild-type cells and the gene set whose expression is selectively altered by loss of MEKK1 regulation of AP-1 composition. Aim 3 will define physiological functions requiring E3 ligase activity and the kinase activity of MEKK1. The targeted knockout of MEKK1 generated in the last funding period provides a null background for add back of MEKK1 wild-type and mutant proteins. To this end, we have developed sensitive assays to measure MEKK1 function both in vitro and in vivo. Cumulatively, this work represents a mechanistic systems analysis of the function and regulation of an important MAP3K, MEKK1, which is critical in the control of homeostasis. MEKK1 has a unique position among the MAP3Ks and our discovery that MEKK1 regulates composition of AP-1 not just AP-1 activity, is a paradigm shift in how MAP3Ks regulate cellular physiology for the control of complex biological responses. PUBLIC HEALTH RELEVANCE: Dysregulation of the MAPK network is associated with many diseases including cancer, inflammation and metabolic disorders. Defining the global control not just of MAPK activity but protein degradation and gene expression by MEKK1 will identify novel therapeutic strategies for treating disease.